My laboratory has utilized both genetic and molecular approaches to study the initiation of cellular proliferation. (1) 3T3 mutants unable to mount a mitogenic response to epidermal growth factor (EGF) and 3T3 mutants unable to proliferate in response to the mitogen/tumor promoter tetradecanoyl phorbol acetate (TPA) have been isolated and characterized. (2) Mutants resistant to the toxic action of EGF coupled to the A-chain of ricin toxin have been selected and characterized. (3) Seven cDNAs, called TIS (TPA Induced Sequences) genes, that are rapidly and transiently induced in response to TPA have modulators (TIS28/c-fos), secreted proteins (TIS7), and proteins of as yet unknown function (TIS10,TIS11, TIS21). The TIS genes can also be induced by other ligands, in a variety of cell-types. SUrprisingly, while no TIS gene is induced only by TPA, we have observed cell-type specific restriction of expression for several TIS genes. Our goals for the next five years include characterization of the functions of the TIS genes, as well as the range and basis of the cell-type restriction of their expression. FUnctional studies will include antibody localization of the TIS proteins, identification of genes whose expression is regulated by the DNA-binding TIS proteins TIS1 and TIS8, the consequences of overproduction and aberrant production of TIS proteins, and the consequences of inhibiting TIS gene expression, using antisense oligonucleotides somatic cell hybrids, transfected reporter genes fused to regulatory regions of the TIS genes, gel-retardation analyses, and chromatin structure probes. Our laboratory (and others) has emphasized isolation and characterization of mitogen-induced genes. However, cellular proliferation is also regulated by inhibitory factors. We will isolate and characterize both primary-response and secondary-response genes induced in response to the proliferation-suppressing protein TGFbeta. Our genetic approach to mitogenesis will continue. We will pursue the biochemical and molecular defects we have identified in our TPA nonproliferative 3T3 mutants. We will also isolate EGF + FGF "double mitogen" nonproliferative mutants, defective in essential post-receptor steps in the proliferative response to these polypeptide growth factors.